Anti-mH2A1 antibody [14G7]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-mH2A1 antibody [14G7] (AB91528)

ab91528 staining mH2A1 in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab91528 at 1µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 594) at 1/1000 dilution (shown in pseudocolour magenta). Nuclear DNA was labelled with DAPI (shown in blue). Also suitable in cells fixed with 4% paraformaldehyde (10 min).Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• WB

Project8335****

Western blot - Anti-mH2A1 antibody [14G7] (AB91528)

All lanes:

Western blot - Anti-mH2A1 antibody [14G7] (ab91528) at 10 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 20 µg

Lane 2:

HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate at 20 µg

Lane 3:

MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate at 20 µg

Secondary

All lanes:

Goat polyclonal Secondary Antibody to Mouse IgG - H&L (HRP), pre-adsorbed at 1/3000 dilution

Predicted band size: 40 kDa

Observed band size: 14 kDa,18 kDa,40 kDa

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Exposure time: 2min

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-mH2A1 antibody [14G7] (AB91528)

Overlay histogram showing HeLa cells stained with ab91528 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab91528, 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

• ChIP

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ChIP - Anti-mH2A1 antibody [14G7] (AB91528)

Chromatin was prepared from SK-OV-3 cells according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min.
The ChIP was performed with 25 µg of chromatin, 5 µg of ab91528 (red), or 5 µg of mouse normal IgG1 ab18443 (gray) and 25 µl of Protein A/G Dyna beads. The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci).
Primers and probes are commercial primers from Paper PMID : 22589551
*http : //www.abcam.com/resources?keywords=X%20ChIP%20protocol

• WB

Lab

Western blot - Anti-mH2A1 antibody [14G7] (AB91528)

Lane 1 : Wild-type HAP1 whole cell lysate (20 μg)
Lane 2 : mH2A1 knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HeLa whole cell lysate (20 μg)
Lane 4 : HepG2 whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab91528 observed at 40 kDa. Red - loading control, ab176560, observed at 50 kDa.

ab91528 was shown to specifically recognize mH2A1 in wild-type HAP1 cells. No band was observed when mH2A1 knockout cells were examined. Wild-type and mH2A1 knockout samples were subjected to SDS-PAGE. ab91528 and ab176560 (Rabbit anti-alpha Tubulin loading control) were incubated overnight at 4°C at 10 μg/ml and 1/10,000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-mH2A1 antibody [14G7] (ab91528)

Predicted band size: 40 kDa

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